Generation Of A Mesenchymal Stromal Cell Bank From The Pooled Mononuclear Cells Of Multiple Bone Marrow Donors

ABSTRACT

The present invention pertains to an improved mesenchymal stromal cell (MSC) preparation and a method for producing the same. The invention provides a new strategy to isolate MSC from bone marrow mononuclear cells (BM-MNCs) by pooling BM-MNCs of multiple unrelated (third-party) bone marrow donors. The MSC preparation manufactured in accordance with the methodology of the invention is characterized by a stable proliferative capability and an increased immunosuppressive potential when compared to individual donor MSC preparations or a pool of individual MSCs generated from multiple donors. The MSCs prepared ac- cording to the invention are particularly useful for medical applications such as the treatment of graft-versus-host disesase (GvHD) in recipients with hematopoietic stem cell transplants, patients with autoimmune disorders and as a cell-based therapy in regenerative medicine.

FIELD OF THE INVENTION

The present invention pertains to an improved mesenchymal stromal cell(MSC) preparation and a method for producing the same. The inventionprovides a new strategy to isolate MSC from bone marrow mononuclearcells (BM-MNCs) by pooling BM-MNCs of multiple unrelated (third-party)bone marrow donors. The MSC preparation manufactured in accordance withthe methodology of the invention is characterized by a stableproliferative capability and an increased immunosuppressive potentialwhen compared to individual donor MSC preparations or a pool ofindividual MSCs generated from multiple donors. The MSCs preparedaccording to the invention are particularly useful for medicalapplications such as the treatment of graft-versus-host disesase (GvHD)in recipients with hematopoietic stem cell transplants, patients withautoimmune disorders and as a cell-based therapy in regenerativemedicine.

DESCRIPTION

Mesenchymal stromal cells (MSCs) since their discovery in the 1970s byFriedenstein et al. have been extensively investigated concerning theirimmunomodulatory and regenerative potential both in vitro and in vivo.In the last decade, considerable progress has been made in elucidatingthe pleiotropic function of MSCs despite the lack of a unique cellsurface marker for their identification and prospective isolation. Inorder to allow for a comparison of the effect of clinically used MSCsfrom different manufacturers, the International Society for CellularTherapy proposed a set of phenotypic and functional criteria to defineMSCs. The absence of HLA-class II antigens and co-stimulatory moleculeson the surface of MSCs, paracrine secretion of a vast array of moleculeswith immunomodulatory potential, as well as the ease of theirprospective isolation from many tissues, makes them a very attractivesource for cell-based therapeutic strategies in a wide range of clinicalconditions such as tissue injuries, inflammation processes andautoimmune disorders. However, for all these clinical applications thereis a need of having at the right moment a large number of the“off-the-shelf” MSCs.

To date, the majority of clinical research was performed usingmesenchymal stromal cells (MSCs) generated from a single bone marrowdonor. As the effects of MSC preparations vary markedly from donor todonor, the results obtained from these studies were to a large extentvery heterogeneous. In addition, the inventors and others havedemonstrated that MSCs exhibit not only a vast donor-to-donor but alsointrapopulation heterogeneity at the clonal level. This remarkableheterogeneity poses the major obstacle in the development of clinicalmanufacturing protocols, which could be used to reproducibly generateMSC-products with an equivalent therapeutic potency.

An isolation process for human mesenchymal stromal cells from bonemarrow samples is described in U.S. Pat. No. 5,486,359. An antibodybinding to mesenchymal stromal cells was developed in order to purifyMSCs from bone marrow aspirates or ground bone material. The bone marrowsamples are separated via a Ficoll gradient and the low density fractionis used for stem cell isolation. U.S. Pat. No. 5,486,359 cultured thecells in normal medium for 1 day on tissue culture plastic to allow stemcells to adhere. Thereafter, the medium is exchanged and the cells arecultured until confluent with medium exchanges every 4 days to obtainthe MSCs.

WO 2012/048093 discloses the isolation of bone marrow derived MSCs fromsingle donors. WO 2012/048093 teaches that MSC preparations derived froma single donor can be expanded to clinical scale preparations.

Pooling of MSC preparations after their generation from the individualbone marrow mono-nuclear cell fractions of unrelated bone marrow donorsor bone samples is known to be possible for the treatment oflife-threatening haemorrhage in a patient with myelofibrosis whounderwent allogeneic hematopoietic stem cell transplantation (O Ringdénand K LeBlanc, Bone Marrow Transplantation, 2011; 46:1158-1160). In thisstudy, clinical-grade MSC preparations derived from two different donorsseparately were combined to increase the likelihood of response.

In view of the above described prior art, a continued need in the clinicexists to prepare clinical grade MSC preparations with a predictableproliferation and immunosuppressive potential and minimal batch-to-batchvariability. Therefore, the problem the present invention seeks to solveis to provide a process for the production/isolation of MSCs from bonemarrow samples with improved characteristics.

The above problem is solved in a first aspect by a mesenchymal stromalcell (MSC) preparation, comprising MSCs isolated from bone marrowmononuclear cells (BM-MNC) characterized in that said MSC preparation ishTERT negative and polygenic. In preferred embodiments said MSC aremammalian, preferably human MSC.

The term “monogenic” when used in context to describe a sample orcomposition of cells refers to these cells originating from a commonsource or having the same genetic background. The term “polygenic” onthe other hand refers to a composition of cells originating fromdifferent sources and having different genetic backgrounds. In contextof the present invention a “polygenic MSC preparation” is a compositioncomprising MSCs having distinct genetic backgrounds, for example MSCswhich originate from at least two genetically distinct bone marrowdonors.

In the context of the herein described invention the terms “mesenchymalstromal cells” and “mesenchymal stem cells” shall be understood to besynonymous descriptions of the same multipotent cell fraction isolatedfrom bone marrow samples.

In order to minimize the inter-donor variability as to theirallosuppressive potential the inventors developed a unique three-steptechnique for the establishment of a GMP-compliant, serum-freeMSC-Master Cell Bank from the pooled bone marrow mononuclear cells(BM-MNCs) of 8 third-party healthy donors: (i) isolation of mixedpolygenic and cryopreservation of individual BM-MNCs from 8 third-partyhealthy donors in full agreement with the approval issued by the localEthics Committee and Declaration of Helsinki, (ii) generation of MSCsfrom pooled BM-MNCs after thawing and cryopreservation in vials and(iii) thawing of MSC samples for their serum-free expansion andgeneration of “off-the-shelf” clinical-scale doses. In this manner, theinventors developed a surprisingly effective protocol for generation ofclinical-grade MSC preparations with a constantly higher allosuppressivepotential and constant proliferative capability, compared to MSCsgenerated from individual bone marrow donors. Therefore, this protocolprovides clinical researchers with clinical-grade MSCs of a consistentquality for the treatment of graft-versus-host disease and otherinflammatory disorders. Despite considerable up-front costs, thisprotocol ensures consistency, reproducibility, and reliability inimmunosuppressive performance of clinical-grade MSCs.

Yet another embodiment pertains to a MSC preparation of the inventionwhich is further characterized in that said MSCs are TERT negative. TERTis the telomerase reverse transcriptase (abbreviated to TERT, or hTERTin humans), which is a catalytic subunit of the enzyme telomerase,which, together with the telomerase RNA component (TERC), comprises themost important unit of the telomerase complex, necessary for maintainingproliferation capability of immortalized cells. The MSC preparation ofthe present invention is shown to comprise MSCs that are not immortal,which is in line with the absent hTERT expression.

One additional preferred embodiment of the invention is a MSCpreparation as described herein, wherein said MSC preparation comprises

-   -   (a) At least 80%, preferably at least 95% CD73+ cells, most        preferably at least 98%, and/or    -   (b) At least 80%, preferably at least 95% CD90+ cells, most        preferably at least 98%, and/or    -   (c) At least 80%, preferably at least 95% CD105+ cells, most        preferably at least 98%, and/or    -   (d) At least 80%, preferably at least 95% HLA-class I+ cells,        most preferably at least 98%, and/or    -   (e) Less than 10%, preferably less than 1% CD45+ cells, most        preferably less than 0.1%, and/or    -   (f) Less than 10%, preferably less than 1% CD14+ cells, most        preferably less than 0.5%, and/or    -   (g) Less than 10%, preferably less than 1% CD34+ cells, most        preferably less than 0.5%, and/or    -   (h) Less than 10%, preferably less than 5% HLA-DR+ cells, most        preferably less than 1%.

In one additional embodiment the MSC preparation comprises

-   -   (a) At least 80%, preferably at least 95% CD73+ cells, most        preferably at least 98%, and    -   (b) At least 80%, preferably at least 95% CD90+ cells, most        preferably at least 98%, and    -   (c) At least 80%, preferably at least 95% CD105+ cells, most        preferably at least 98%.

Additionally the MSC preparation of the invention may comprise

-   -   (e) Less than 10%, preferably less than 1% CD45+ cells, most        preferably less than 0.1%, and    -   (f) Less than 10%, preferably less than 1% CD14+ cells, most        preferably less than 0.5%, and    -   (g) Less than 10%, preferably less than 1% CD34+ cells, most        preferably less than 0.5%.

The problem of the present invention is furthermore solved by anin-vitro method for the isolation of mesenchymal stromal cells, themethod comprising: pooling bone marrow samples obtained from at leasttwo genetically distinct donors to obtain a sample cell-pool, andthereafter isolating mesenchymal stromal cells from said samplecell-pool.

The process for the preparation or isolation of MSC from bone marrowsamples in accordance with the present invention involves a step ofpooling bone marrow, or a mononuclear cell fraction derived from bonemarrow, from genetically distinct donors. Therefore, at least twogenetically distinct bone marrow samples, or genetically distinctBM-MNCs fractions, are pooled in the method of the present invention.Importantly, the inventive method comprises the pooling of geneticallydistinct BM samples at a stage where the BM samples contain still amixture of different cell types. Therefore, preferably the pooling ofgenetically distinct BM-MNC samples is done before the MSC fraction ispurified or expanded from said samples. The pooling of the cells beforethe isolation of MSC yielded into MSC preparations with a surprisinglyimproved allo suppressive potential.

In one embodiment the method according to the invention comprises thesteps of:

-   -   (a) Providing a number of bone marrow samples obtained from at        least two genetically distinct donors,    -   (b) Pooling said bone marrow samples to obtain a sample        cell-pool,    -   (c) Optionally, culturing said sample cell-pool, and    -   (d) Isolating from said sample cell-pool obtained in step (b)        said mesenchymal stromal cells.

In one preferred embodiment the method comprises the steps of: Providinga number of bone marrow samples obtained from at least two geneticallydistinct donors, isolating the mononuclear cell fraction from bonemarrow samples, pooling said bone marrow mononuclear cell samples toobtain a BM-MNC-pool, optionally, culturing said BM-MNC-pool, andisolating from said BM-MNC-pool said mesenchymal stromal cells. Ofcourse all additional special variations of the inventive method asdescribed herein above and below are equally preferable embodiments ofthis methodology which includes a step of isolating BM-MNCs from bonemarrow samples.

The term “sample cell-pool” in context of the invention shall refer to amixture of bone marrow derived cells with different genetic background.Therefore, the sample cell-pool of the invention is polygenic. Mostpreferably a sample-cell pool in accordance with the invention ischaracterized in that MSCs are present only to a minor percentage ofpreferably less than 80%, preferably 70%, 60%, 50%, 40%, 30%, 20% andmost preferably less than 10%, even more preferably less than 5%, lessthan 1%, most preferably less than 0.1%.

The term “genetically distinct” as used herein, indicates that at leastone difference at the genomic level exists between bone marrowdonors/subjects. Bone marrow can be collected from 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more donors. Most preferredis the use of 4 to 8 different donor samples.

A “bone marrow sample” in accordance with the present invention mayeither be a bone marrow aspirate or from bone pieces, such as cancellousbone pieces. In a preferred embodiment a bone marrow sample is a bonemarrow aspirate. How to collect a bone marrow sample is known to theskilled artisan. Said bone marrow samples of the invention in onepreferred embodiment comprise a mixture of different cell types. Forexample each of said bone marrow samples comprises at least onenon-adherent cell fraction and at least one adherent cell fraction. Inone particular preferred embodiment said bone marrow sample is a bonemarrow mononuclear cell (BM-MNC) sample or fraction. BM-MNCs areobtained from bone marrow.

In context of the present invention a “bone marrow mononuclear cellfraction” (also referred to herein as “BM-MNCs”), contains B, T and NKlymphocytes, early myeloid cells, and a very low number of endothelialprogenitors, hematopoietic stem/progenitor and/or mesenchymal stromalcells.

In a preferred embodiment of the invention said bone marrow sample is amammalian bone marrow sample and said mesenchymal stromal cell is amammalian mesenchymal stromal cell. More preferred is that said bonemarrow sample is a human bone marrow sample and said mesenchymal stromalcell is a human mesenchymal stromal cell.

Yet another embodiment of the invention pertains to the afore describedprocess for the isolation of MSCs, the method further comprising a step(a′) of extracting bone marrow mononuclear cells (BM-MNC) from each ofsaid bone marrow samples to obtain BM-MNC-samples, and wherein in step(b), said BM-MNC-samples are pooled to obtain said sample cell-pool. Asmentioned above, the BM-MNC fraction/sample contains only a small numberof MSC. Therefore, in one preferred embodiment of the invention saidBM-MNC samples to be pooled comprise a percentage of mesenchymal stromalcell per total cells in said sample of less than 80%, preferably 70%,60%, 50%, 40%, 30%, 20% and most preferably less than 10%, even morepreferably less than 5%, less than 1%, most preferably less than 0.1%.

In context of the herein described invention, in all of its embodimentsand aspects, it is particularly preferred that said bone marrow samples(or BM-MNC samples) are obtained from at least three, more preferably atleast four, more preferably at least five, more preferably at least six,more preferably at least seven, and most preferably at least eightgenetically distinct donors. In other words, the MSC cell preparationand method for its production in one particularly preferred embodimentcomprises the pooling of at least three, more preferably at least four,more preferably at least five, more preferably at least six, morepreferably at least seven, and most preferably at least eightgenetically distinct cellular samples.

Methods for the isolation of BM-MNCs from a bone marrow sample, inparticular bone marrow aspirate, are well known to the person of skill.However, in one embodiment of the present invention it is preferred thatBM-MNCs are isolated from a bone marrow sample using a cell densityseparation such as Ficoll gradient.

Once pooled, the obtained sample cell-pool of the invention is used toisolate and purify the MSCs. Methods for the isolation of MSCs from bonemarrow samples or BM-MNCs are also well known in the art. A preferredmethod of the invention is the separation of adherent from non-adherentcells by simply removing floating cells by aspirating the cell culturemedium from the culture container at regular intervals. By exchangingthe medium multiple times the fraction of non-adherent cells isconstantly reduced, whereas the adherent MSC-fraction continues to growuntil confluency. These adherent cells are MSCs.

Thus, preferred is in one embodiment that step (d) of the method of theinvention comprises a step of removing at least once the non-adherentcells from a culture of said sample cell-pool; or wherein afterculturing said sample cell-pool, at least one detectable surface markeror antibody is used for the purification of said MSCs.

Additionally the method of the invention may provide a further step ofeither storing said isolated mesenchymal stromal cells, or expandingsaid isolated mesenchymal stromal cells.

In one additional aspect the invention includes a method for theproduction of clinical-grade MSC preparations. This method includes theaforementioned method steps for isolating MSCs, but furthermore includesthe expansion of the isolated MSCs until receiving an amount of MSCapplicable in the clinic. The expansion of the MSCs of the invention mayeither follow immediately after isolating the MSCs, or alternatively theisolated MSCs were stored via cryopreservation, by thawing an aliquot ofcells for starting the expansion process. How MSCs are expanded is knownin the art, and as an example explained in the example section of thepresent application.

Another aspect of the present invention pertains to a cellularcomposition comprising bone marrow samples from at least two geneticallydistinct bone marrow donors. Alternatively the cellular composition ofthe invention may comprise BM-MNCs from at least two geneticallydistinct bone marrow donors. Therefore, the cellular composition of theinvention is preferably polygenic.

In one embodiment the cellular composition of the invention isobtainable by pooling at least two monogenic and genetically distinctbone marrow samples before isolating and/or expanding a stem cellfraction contained in said bone marrow samples. Thereby, via pooling,the cellular composition becomes polygenic.

In one preferred embodiment said bone marrow samples are bone marrowmononuclear cell samples.

One embodiment of this aspect pertains to a cellular composition of theinvention, obtainable by a method comprising the steps

-   -   (a) Obtaining at least two bone marrow samples, each one of        those from a genetically distinct bone marrow donor,    -   (b) Isolating from each of said bone marrow samples the bone        marrow mononuclear cell fraction to obtain monogenic bone marrow        mononuclear cell samples, and    -   (c) Pooling said monogenic bone marrow mononuclear cell samples        obtained from each bone marrow sample to obtain the polygenic        cellular composition.

It is understood that in context of the present invention a “bone marrowmononuclear cell fraction” comprises the standard cellular compositionknown for this cell fraction derived from bone marrow. In this regard itis preferred that said monogenic mononuclear cell samples are pooledbefore performing a further step of cellular purification or expansion,preferably before isolating or purifying any MSCs therefrom.

Yet another aspect of the invention provides a use of a mixture of bonemarrow mononuclear cells obtained from at least two genetically distinctbone marrow donors in a method of isolating mesenchymal stromal cells.

Further provided is the aspect of a use of a cellular composition asdescribed herein before in a method of purification/isolation ofmesenchymal stromal cells.

The problem of the invention is also solved by a mesenchymal stromalcell obtainable by a method for isolating/purifying MSCs as describedherein above.

The herein described MSC preparations or MSCs are preferably for use inmedicine. MSCs are generally used in a wide variety of medicalapplications. Without intending to be restricted to the followingexamples, the MSCs of the invention are preferably for use in thetreatment of autoimmune diseases such as multiple sclerosis, Type 1diabetes, rheumatoid arthritis, uveitis, autoimmune thyroid disease,inflammatory bowel disease (IBD), scleroderma, Graves' Disease, lupus,Crohn's disease, autoimmune lymphoproliferative disease (ALPS),demyelinating disease, autoimmune encephalomyelitis, autoimmunegastritis (AIG), and autoimmune glomerular diseases. Particularly, theMSC preparation of the invention is used in the treatment ofgraft-versus-host disease (GVHD).

However, in context of the herein described invention, the MSCpreparations are useful in any regenerative or autoimmune disease,preferably transient, relapsing or remitting. Other clinicalapplications of the inventive MSC preparations are in wound healing,corneal ulcer, stroke, or for facilitating of engraftment in allogeneicstem cell transplantation.

A cell therapy involving MSC administration is based, for example, onthe following steps: harvest of MSC-containing tissue (bone marrow),isolate and expand MSCs in accordance with the herein described methods,and administer the MSCs to the subject/patient, with or withoutbiochemical or genetic manipulation.

In one aspect, the invention provides a method of treating a subject inneed thereof comprising the step of administering a therapeutic dose ofan MSC preparation produced in accordance with the present invention.

A therapeutic dose for an autoimmune disease or graft-versus-hostdisease can contain about 1×10⁵ cells/kg to about 1×10⁷ cells/kg. Inanother embodiment, a therapeutic dose is about 1×10⁶ cells/kg to about5×10⁶ cells/kg. In another embodiment, a therapeutic dose is about 2×10⁶cells/kg to about 8×10⁶ cells/kg. In another embodiment, a therapeuticdose is about 2×10⁶ cells/kg or about 2×10⁶±about 10%, about 20%, orabout 30% cells/kg. In another embodiment, a therapeutic dose is about8×10⁶ cells/kg or about 8×10⁶±about 10%, about 20%, or about 30%cells/kg, and include any amounts or ranges there between. Given an MSCpreparation of the present invention, the number of mesenchymal stromalcells to be administered is dependent upon a variety of factors,including the age, weight, and sex of the patient, the disease to betreated, and the extent and severity thereof.

The MSCs of the invention can be administered by a variety ofprocedures. MSCs can be administered systemically, such as byintravenous, intraarterial, or intraperitoneal administration. Themesenchymal stromal cells can be administered by direct injection to anorgan or tissue in need thereof. The mesenchymal stromal cells can beapplied topically. The mesenchymal stromal cells can be applied directlyto a tissue in need thereof during a surgical procedure.

The mesenchymal stromal cells, in accordance with the present invention,can be employed in the treatment, alleviation, or prevention of anydisease or disorder which can be alleviated, treated, or preventedthrough angiogenesis. Thus, for example, the mesenchymal stromal cellscan be administered to an animal to treat blocked arteries, includingthose in the extremities, i.e., arms, legs, hands, and feet, as well asthe neck or in various organs. For example, the mesenchymal stromalcells can be used to treat blocked arteries which supply the brain,thereby treating or preventing stroke. Also, the mesenchymal stromalcells can be used to treat blood vessels in embryonic and postnatalcorneas and can be used to provide glomerular structuring. In anotherembodiment, the mesenchymal stromal cells can be employed in thetreatment of wounds, both internal and external, as well as thetreatment of dermal ulcers found in the feet, hands, legs or arms,including, but not limited to, dermal ulcers caused by diseases such asdiabetes and sickle cell anemia.

Furthermore, because angiogenesis is involved in embryo implantation andplacenta formation, the mesenchymal stromal cells can be employed topromote embryo implantation and prevent miscarriage.

In addition, the mesenchymal stromal cells can be administered to anunborn subject, including humans, to promote the development of thevasculature in the unborn subject.

In another embodiment, the mesenchymal stromal cells can be administeredto a subject, born or unborn, in order to promote cartilage resorptionand bone formation, as well as promote correct growth platemorphogenesis.

The mesenchymal stromal cells can be genetically engineered with one ormore polynucleotides encoding a therapeutic agent. The polynucleotidescan be delivered to the mesenchymal stromal cells via an appropriateexpression vehicle. Expression vehicles which can be employed togenetically engineer the mesenchymal stromal cells include, but are notlimited to, retroviral vectors, adenoviral vectors, and adeno-associatedvirus vectors. The MSCs of the invention can for example be geneticallyengineered to overexpress TERT, and thereby to immortalize the cells.

Also, the MSC preparation of the invention or the mesenchymal stromalcell can be for use in stem cell transplantation.

Further provided is a use of the MSC preparation or MSCs of theinvention in the production of bone replacement material.

Another aspect of the invention is a method for the production of amedicament comprising mesenchymal stromal cells, comprising the methodsteps according to any of the herein described methods for theisolation/purification of MSCs.

In another embodiment, the MSCs of the present invention or producedwith the methods of the invention can differentiate into osteoblasts,adipocytes and/or chondrocytes under appropriate culture conditions,such as the respective cell differentiation inducing conditions, forexample cultured with the appropriate inducing medium, such asosteogenic, adipogenic and chondrogenic induction medium, respectively.In one embodiment, the appropriate culture conditions and appropriateinducing media are those specified in the Examples. In anotherembodiment, examples of suitable conditions and media are disclosed inAubin J E. Osteoprogenitor cell frequency in rat bone marrow stromalpopulations: role for heterotypic cellcell interactions in osteoblastdifferentiation. J Cell Biochem. (1999) 72(3):396-410 (forosteogenesis); Falconi D, Oizumi K, Aubin J E. Leukemia inhibitoryfactor influences the fate choice of mesenchymal progenitor cells. StemCells. (2007) 25(2):305-312 (for adipogenesis); and Zhang S, Uchida S,Inoue T, Chan M, Mockler E, Aubin J E. Side population (SP) cellsisolated from fetal rat calvaria are enriched for bone, cartilage,adipose tissue and neural progenitors. Bone. (2006) 38(5):662-670 (forchondrogenesis).

In one aspect of the invention, the invention provides a kit forconducting the method of the invention comprising one or more of thefollowing: culture medium, and instructions for using same. In anotherembodiment the invention can provide a kit comprising a sample ofisolated mesenchymal stromal cells of the present invention andoptionally culture medium and or instructions for use in experimentsand/or in transplantation.

The mesenchymal stromal cell (MSC) preparation as described herein isfurthermore characterized in that it comprises MSCs isolated from bonemarrow mononuclear cells (BM-MNC), that are polygenic, and having atleast one of the following characteristics:

-   -   (a) an increased p21 expression compared to the p21 expression        in monogenic MSCs (generated from single donors),    -   (b) a decreased p53 expression compared to the p53 expression in        monogenic MSCs (generated from single donors), and/or    -   (c) a decreased c-myc expression compared to the c-myc        expression in monogenic MSCs (generated from single donors).

These characteristics might be used as alternative or additionalstructural features distinguishing the described MSC preparation of theinvention from state of the art preparations. In one embodiment the MSCpreparation of the invention comprises MSCs with an increased p21expression compared to the p21 expression in monogenic MSCs (generatedfrom single donors). The MSCs of the invention may further oralternatively characterized by a decreased p53 expression compared tothe p53 expression in monogenic MSCs (generated from single donors).

The present invention in one embodiment relates to a MSC preparation,wherein said MSCs are further or alternatively characterized by adecreased c-myc expression compared to the c-myc expression in monogenicMSCs (generated from single donors).

In specific embodiments said increase in p21 expression is at least2-fold, preferably at least 3 fold, more preferably at least 4 fold;and/or wherein said decrease in p53 expression is at least 10-fold,preferably at least 20 fold; and/or wherein said decrease in c-mycexpression is at least 10-fold, preferably at least 20 fold, and mostpreferably not detectable.

Preferred MSC preparations of the invention comprise MSCs with all ofthe aforementioned characteristics (a) to (c). The isolated MSCs of theinvention comprised at least one human MSC with a chromosomaltranslocation between chromosomes 5 and 9, which may be used as anadditional characteristic of the MSCs of the invention. Preferably MSCsare characterized by (a) and (b), (a) and (c), or (b) and (c). Mostpreferably said MSCs are characterized by (a), (b) and (c). A MSCpreparation is also preferred, wherein said difference in expression ofp21, p53, c-myc, and/or hTERT is between invented MSCs and monogenicMSCs generated from single donors.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures:

FIG. 1: Collection of bone marrow and separation of bone marrowmononuclear cells. A) Bone marrow was collected from 8 healthythird-party donors in general anaesthesia by bilateral aspiration fromthe iliac crest. B) Bone marrow samples were collected in bags andanti-coagulated with 7-12% ACD-A and 7-12 i.U. of heparin per ml ofmarrow aspirate. C) Bone marrow mononuclear cells were enriched frombone marrow aspirate by Ficoll (GE Healthcare, Munich, Germany) densitycentrifugation using the Sepax II NeatCell process (Biosafe,Switzerland). D) BM-MNCs were washed twice and resuspended in thecryomedium consisting of 10% DMSO/5% HSA/X-vivo. E) Bags containingBM-MNCs from each bone marrow donor in cryomedium were frozen in thevapour phase of liquid nitrogen until use.

FIG. 2: Generation of the MSC-bank from bone marrow mononuclear cells.A) Bags containing bone marrow mononuclear cells of each donor werethawed at +37° C. and after washing twice with medium they were pooledin a definite volume of DMEM supplemented with 5% platelet lysate. Allcells were plated in one 1-CellStack and eleven 2-CellStack plates B)After 72 hours the non-adherent fraction was removed and the adherentcells were further cultured in DMEM supplemented with 5% PL for another11 days by changing medium every three days. Once the MSCs appeared andgrew to a confluence of 80% MSCs were detached using trypsin (TrypLE)and after washing them with medium cell pellets were resuspended incryomedium consisting of 10% DMSO/5% HSA/DMEM. C) MSCs were frozen in210 cryovials, each containing 1.5×10⁶ MSCs of passage Pl. The inventorsdesignated this set of vials with MSCs as a MSC-bank.

FIG. 3: Generation of MSC-clinical end products A) Three randomly-chosencryopreserved vials with MSCs were thawed 6-8 weeks after their initialcryopreservation B) They were plated in one 1-CellStack (636 cm²) andcultured for 6-7 days in DMEM containing 10% platelet lysate. The mediumwas changed every three days. C) On day 6 or 7 (according to theirconfluent growth) MSCs (the end of P1) were detached by trypsin, washedand plated in eight 2-CellStacks at a cell concentration of 2×10³ MSCs/1cm2 and expanded for another week. Medium was changed every 3-4 days andat the end of the week (the end of P2) MSCs were detached by trypsin,washed twice and the cell number was counted. These MSCs werecryopreserved and designated as MSC clinical product, which after 2-3weeks underwent validation concerning their proliferative,differentiation and allo suppressive potential.

FIG. 4: Phenotype of MSCs and their differentiation potential. A) MSCsgenerated by expansion of the aliquots from MSC-bank at the end ofpassage 2 were labelled with mouse anti-human antibodies conjugated tofluorochromes as presented in the table 1. B) Culture of MSCs in thetissue-specific culture media induced their differentiation intoadipocytes and osteoblasts.

FIG. 5: Growth kinetics of MSCs from individual donors and the MSC-endproducts. A) The initial number of 4.4×10⁴ MSCs from each bone marrowdonor was expanded for one passage (from the start to the end of P2). Atthe same time, MSCs of all 8 donors were pooled and expanded from thestart till the end of P2 (MSC-Pool) as well as 4 aliquots from theMSC-Bank. At the end of passage 2 the MSCs were trypsinized and theirnumber was calculated; ns=not significant B) Ten MSC-cryovials ofMSC-bank were thawed and expanded over two passages in order to assesstheir proliferation potential. Mean cell number of all expanded vials atthe end of passage 2 was 5.3×10⁸±5×10⁷ MSCs. C) MSCs showedapproximately 4 population doublings (PDs) per passage, therefore thecumulative number of PDs (CPD) was 8.7±0.4 PDs. D) To demonstrate thatthe MSC-end products are not immortal cells, the inventors assessedtheir growth kinetics for 12 passages and estimated the number of PDs.As it is shown in the figure from passage 9-12 these MSCs were not ableto even duplicate themselves (n=3).

FIG. 6: Allosuppressive potential of MSCs generated from individualdonors and MSC-end products. A) MSCs of passage 0 from 8 individualdonors as well as the MSC-Pool that was generated by pooling the MSCs of8 donors before expansion (MSC-Pool), and one MSC-end product (MSC-140)were expanded to the end of passage 2. Thereafter, the MSCs weretrypsinized, washed twice in the medium and after determination of thecell counts and viability by trypan blue they were used to estimatetheir allosuppressive potential in MLR-assay. On day 6 BrdU was offeredto the cells and next day the assay was performed in order to evaluatethe inhibition of proliferation of allogeneic blood mononuclear cellsfrom two HLA-disparate donors. B) Six MSC-end products (clinical doses)were thawed and after washing the cells they were directly used for theMLR-assay. The aim of these experiments was to find out whether thawedMSC-end products, as they are given to the patients, are able tosuppress the allogen-induced proliferation of mononuclear cells from twoallogeneic MNC donors.

FIG. 7: Genetic characterization of the clinical-scale MSC-end productA) Normal karyogram of the clinical-grade MSC-end products at the end ofpassage 2. B) Interphase nuclei after two-color hybridization of probeset 5p15 (green) and 5q35 (red) identified a normal diploid pattern forthe chromosome 5. C) Interphase nuclei after three-color hybridizationof a MYC break apart probe showed in almost all cells two normal fusionsignals. D) Number of MSCs with normal diploid and aneuploid patternafter two-color hybridization of probe set 5p15 and 5q35. Total numberof analyzed MSCs was 396. E) Number of MSCs with normal diploid andaneuploid pattern after three-color hybridization of a MYC break apartprobe for chromosome 8q24. Total number of analyzed MSCs was 356.

FIG. 8: Expression of transforming genes and chimeric analysis of theMSC-end products A) RT-PCR analysis of genes involved in the celltransformation in 3 clinical-scale MSC-end products. Total RNA wasisolated from three MSC-end products and MSCs from one donor (control).After transcription into cDNA it was used to quantify expression of p21,p53 and c-myc by PCR. B) STR-PCR analysis of the clinical-scale MSC-endproduct. From the MSCs of the clinical-grade MSC-end product DNA wasisolated, which then was used to evaluate specific STR-regions found onnuclear DNA. All eight donors' genotype was represented in the MSC-endproduct generated from pooled MNCs.

EXAMPLES

Materials and Methods

Raw Material Collection

Bone marrow was aspirated in general anaesthesia by bilateral aspirationfrom the iliac crest. Marrow was anti-coagulated with 7-12% ACD-A and7-12 i.U. of heparin per ml of marrow aspirate.

Infectious Disease Testing

The infectious disease marker panel exceeded minimal requirements ofJACIE and the German Stem Cell Act. Thus as part of the donor work-up,evidence of seronegativity for HiV1/2, anti-HBc, HBsAg, anti-HCV,anti-HTLV1/2 (IgM and IgG for all), anti-Hepatitis A IgM,anti-Toxoplasma IgM, anti-EBV IgM, anti-CMV IgM and TPHA, as well asnegativity by NAT for HiV, HAV, HBV, HCV and ParvoB 19 was sought; testsfor HiV1/2, anti-HBc, HBsAg, anti-HCV, CMV, TPHA and the described NATwere repeated on the day of marrow donation. All donors met thecriteria. Moreover, since CMV is a cell-resident virus, negativity forCMV genome in the bone marrow cell pellet was sought (Dept. of Virology,Goethe University, and Bioreliance, Glasgow, UK).

Processing Facility

All processes were performed under full GMP criteria in the clean roomsuite (class A in B) of the Department of Cellular Therapeutics/CellProcessing (GMP) which is part of the German Red Cross Blood Service andfully embedded into the quality management system thereof, with formalpermission from the state government (manufacturing license acc. to§20b/c (BM collection and testing) and §13 (MSC generation and testing)German Medicines Act).

Bone Marrow Processing

Bone marrow mononuclear cells were enriched from bone marrow aspirate byFicoll (GE Healthcare, Munich, Germany) density centrifugation using theSepax II NeatCell process (Biosafe, Switzerland) as described by themanufacturer. All connections were established by sterile tube welding(TSCD, Terumo, Dusseldorf, Germany), so that an entirely closed processwas used.

Collection of Platelet Concentrates and Generation of Platelet Lysates(PL)

As starting material for platelet lysates one to two day-old buffy coatpool platelets containing approximately 10% plasma in PASIII were used.Platelets were cleared for clinical use in accordance with Germanguidelines for blood products. Up to 4-6 platelet concentrates werepooled to generate one batch of platelet lysate. Platelets werealiquoted in an A in B environment into sterile 50 ml Falcon tubes andimmediately frozen at −80° C. Individual aliquots were thawed after atleast 24h and centrifuged for 10 minutes at 3774×g with brake(acceleration 8, brake 4). The supernatants (platelet lysates) werecollected and subjected to extended release testing, including freedomfrom bacteria (BacTAlert, Biomerieux) and potential to promote theadherence of progenitor cells for MSC and MSC-expansion.

Testing of Platelet Lysates

a) Potential of platelet lysates to promote the adherence of progenitorcells for MSCs Bone marrow mononuclear cells (BM-MNCs) were thawed at37° C., washed twice with DMEM supplemented with 5% PL and after thelast wash the cell pellet was resuspended in the DMEM supplemented with5UI Heparin/m1 and 5% or 10% PL. BM-MNCs were plated at a density1.71×10e5/1 cm2 and incubated for 72 hours at 37° C. with 5% CO₂ andsaturated humidity. The non-adherent cell fraction was removed, whilethe adherent cells were further cultured for another 11 days. Culturemedium was changed every 3-4 days. Once the spindle-shaped cells (MSCs)were confluent at 70-80% they were enzymatically detached by trypsin andtheir number was counted. This procedure was performed with both mediai.e. DMEM supplemented with either 5% PL or 10% PL.

b) Capacity of PLs to Expand MSCs

The specification of platelet lysates was set as IDM-conforming tostandards laid out in the German guidelines, freedom from bacteria,expansion of a cryopreserved aliquot of MSC by at least 2-fold within 7days. Only platelets fulfilling these criteria were used for thegeneration of clinical MSC protocols.

Generation of MSC-Bank and Clinical-Grade End Products

a) Generation of MSC-Bank

Bags containing bone marrow mononuclear cells from each donor werethawed in Plasmatherm at +37° C. They were washed twice with DMEMsupplemented with 5% PL by centrifugation for 10 minutes at 400 g andresuspended at a defined volume of DMEM+5% platelet lysate. After thisstep, cell suspensions from each donor were pooled together and thenumber of cells was counted by a cell counter (Sysmex). Thereafter, cellsuspension pool was plated in eleven 2-CellSTACKs (per one 2-CellSTACK:250×10e6 BM-MNCs/260 ml medium) and in 1 single CellSTACK (per1-CellSTACK: 125×10e6 BM-MNCs/130 ml medium). After 72h the nonadherentcells were removed using medium exchange bags (Macopharma, Langen,Germany) and adherent cells were cultured further for another 11 dayswith DMEM supplemented with 5% PL until the MSCs were 80-90% confluent.In this period the medium was changed every 3-4 days. On day 14 beforedetachment of the MSCs, from each Cell-STACK was taken 5 ml of culturemedium which was pooled in a sterile bottle to be tested for sterilityfor aerobic and anaerobic bacteria and fungi. Thereafter, the cells weredetached using synthetic TrypLE (Life Technologies, Darmstadt, Germany)and after washing them with DMEM+5% PL, the cells were centrifuged for 7minutes at 400×g. Cell pellets were resuspended in defined volume ofmedium and the cells were counted with trypan blue in a hemacytometer.The inventors obtained in total 320×10e6 passage 1 MSCs. Two-millioncells were used for determination of the phenotype by means of flowcytometry. The rest of MSCs was centrifuged for 7 minutes at 400×g.

The cells were resuspended in 5%HSA/DMEM whereby the number of MSCs wasadjusted to 3×10e6 cells/ml. One volume of cell suspension was slowlymixed with one volume of cold freezing medium containing 20%DMSO/5%HSA/DMEM. Therefore, the final concentration of MSCs was 1.5×10e6/mlcell suspension, whereas the final concentration of cryomedium was 10%DMSO/5% HSA/DMEM. The cells were distributed in 210 cryovials (each1.5×10e6 MSCs of passage 1) and then cryopreserved by using a Cryoservecontrolled-rate freezer (Schollkrippen, Germany) according toestablished protocols. The frozen vials were stored in vapour phase ofliquid nitrogen (Tec-Lab, Idstein, Germany). In addition, the rest ofMSCs was mixed with freezing medium and tested for sterility (aerobicand anaerobic bacteria and fungi).

b) Generation of Clinical-Scale MSC-End Products

To generate and validate the clinical-scale MSC-end products, MSC-bankvials were successively thawed per random 6-8 weeks after theircryopreservation. After thawing at 37° C. MSCs were washed in culturemedium containing 10% PL, whereby the cell count viability was assessedusing trypan blue staining in hemocytometer. The cells of one MSC-bankvial was plated in one 1-CellStack (636 cm²) and cultured with DMEMsupplemented with heparin (5 IU/ml medium) and PL 10%(V/V). The mediumwas changed on day 4 and on day 6-7 the cells were detached using TrypLeand then further plated in eight 2-CellStacks (each 1,272 cm²) aspassage two at a density 2×10³ cells/cm². The procedure was repeated asfor passage 1 and on day 6-7 the MSCs were harvested. After washing with0.5% HSA/0.9% NaCl, viable MSCs were counted by trypan blue staining.Further, clinical-scale MSCs were resuspended in cryomedium (0.9% NaClwith 5% HSA and 10% DMSO) as end passage 2 MSCs and distributed in 4-7cryobags containing each 4,2 to 5.5×10e7 MSCs in a volume of 45 mlfreezing medium. Samples were cryopreserved using a Cryoservecontrolled-rate freezer (Schollkrippen, Germany) using establishedprotocols and stored in vapour phase of liquid nitrogen (Tec-Lab,Idstein, Germany).

Quality Control Tests

All tests were performed with thawed MSC-end products without expansion,in a state in that they are administered to the patients for clinicalapplication.

a) Cell Enumeration and Viability of Thawed MSCs

Total cell enumeration was done using a Neubauer chamber hemacytometer.MSC viability was assessed using trypan blue staining.

b) Phenotypic Characterization

Flow cytometric analysis was performed using the ISCT minimal criteriafor MSCs. To determine the phenotype of thawed MSCs the inventorsstained them with following fluoro-chrome-conjugated mouse anti-humanmonoclonal antibodies as presented in table 1.

TABLE 1 Antibodies used for determination of the phenotype of MSCsAntibodies Company Cat. Nr. Clone Isotype IgG1 FITC BioLegend 400109MOPC-21 IgG1 IgG2a FITC BioLegend 400209 MOPC-173 IgG2a IgG1 PEBioLegend 400113 MOPC-21 IgG1 IgG1 PerCP BioLegend 400147 MOPC-21 IgG1CD45 FITC BioLegend 304005 HI30 IgG1 CD34 FITC BioLegend 343603 561IgG2a CD14 FITC BioLegend 325603 HCD14 IgG1 HLA-DR FITC BioLegend 307603L243 IgG2a CD90 FITC BioLegend 328107 5E10 IgG1 CD73 PE BioLegend 344003V B-CD73.3 IaG1 CD105 PE BioLegend 323205 43A3 IgG1 Propidium iodide BDPharmingen 556463 (PI) Solution

d) Evaluation of the Allosuppressive Potential of MSC-End Products

To test the immunosuppressive effect of the MSC-end products on thealloantigen-driven reaction, the inventors used mixed lymphocytereaction (MLR). Peripheral blood mononuclear cells (PB-MNCs) fromhealthy unrelated donors were isolated using a Ficoll-gradient (density1.077, Biochrom KG, Berlin, Germany), washed twice with PBS andresuspended in RPMI-1640 with 10% FBS (Invitrogen). PB-MNCs of 2unrelated donors were cultured in black 96-well plates for six dayseither alone (control group) or mixed with third-party, lethallyirradiated (30 Gy) MSC-end products at an MSC:PB-MNC ratio of 1:1 (1×10⁵MSC:1×10⁵ PB-MNC). The inventors assessed six MSC-end products directlyafter thawing as well as MSCs of all eight donors, whose BM-MNCs werepooled and served as a source for generation of the inventor's masterMSC-bank. All MLRs were performed in triplicates in a 96-well plate. Onday 6, cells were incubated with 5-bromo-2′-deoxyuridine (BrdU) (RocheDiagnostics GmbH, Mannheim, Germany) for 24 h. The following day,relative light units (RLU/s) were measured with a luminometer 1420Multilabel Counter Victor 3 (Perkin Elmer, Rodgau-Jügesheim, Germany).Proliferation levels of PB-MNCs were determined on day 7 using a BrdUassay. The inhibitory effect of MSC-end products on the proliferation ofallogeneic MNCs was calculated as a percentage using the followingformula: 100-[(proliferation of allogeneic PB-MNCs in presence ofMSC/proliferation of PB-MNCs without MSCs)×100].

e) Determination of the Senescence of MSC-End Products In Vitro

To demonstrate that the MSC-end products are not immortal cells, theinventors assessed their growth kinetics over 12 passages. For eachpassage the medium was changed every 3 to 4 days and detachment of MSCsby TrypLE was performed according to their proliferation potential. Tomore precisely estimate their growth kinetics the inventors calculatedthe number of population doublings (PDs) by using the following formula:

PD for each subculture: [log 10(NH)-log 10(NI)/]log 10(2); where NH=cellharvest number, NI=inoculum number of cells.

f) Differentiation Potential of MSC-End Products

To study differentiation potential along adipogenesis and osteogenesisMSC-end products of passage 2 were thawed and directly cultured in theappropriate tissue-specific induction media according to manufacturer'sinstructions (Miltenyi Biotec GmbH).

Adipogenesis

In order to generate adipocytes, the number of thawed MSCs was adjustedto 5×10e4 cells/1 ml of NH AdipoDiff Medium. Then, 1.5 ml of such a cellsuspension were cultured in 35 mm cell culture dishes at 37° C. in anincubator with 5% CO2 and >95% humidity. The medium was changed every 3dday and after 2-3 weeks large vacuoles started to appear. On day 30 theadipocytes were rounded and filled with lipid droplets, which theinventors stained with Oil Red O (Millipore, Schwalbach, Germany), alipophilic red dye.

Osteogenesis

Briefly, concentration of thawed MSCs was adjusted to 3×10e4 cells/1 mlof NH OsteoDiff Medium. Then, 1.5 ml of such a cell suspension werecultured in 35 mm cell culture dishes at 37° C. in an incubator with 5%CO2 and >95% humidity. The medium was changed every 3d day. On day 10the osteoblasts can be identified morphologically by their cuboidalappearance and by their association with newly synthesized bone matrix.These cells are visualized by alkaline phosphatase staining (Sigma,Deisenho fen, Germany), since committed osteogenic cells express highlevels of this enzyme. As a result of this staining the osteoblastsappear as dark purple stained cells. Tissue-specific stainings wereevaluated using an Olympus IX71 microscope equipped with Soft ImagingSystem F-View II camera and cellSens Dimension imaging software.

g) Genetic Analysis of the Clinical-Grade MSC-End Products

RT-PCR analysis of the expression of transforming genes ofclinical-scale MSC-end products. RNA was extracted using the RNeasy minikit (Qiagen) followed by reverse transciption with 1 μg of total RNAusing the Verso cDNA Kit (Thermo Scientific) with random hexanucleotidesaccording to manufacturer's instructions, respectively. For the c-myc.p21, p53 and GAPDH gene real time PCR was performed on an Eppendorfrealplex using the Quanti Tect SYBRE green qPCR master Mix (Qiagen).Detection of hTERT and ABL gene transcription was performed on a BioradMyiQ Cycler using the Absolute qPCR ROX mix (Thermo Scientific).Oligonucleotides were purchased at Eurofins MWG. Primer sequences andPCR conditions except the reaction mix specific activation periods havebeen published in detail elsewhere.

h) Interphase Fluorescence In Situ Hybridization (FISH)

Interphase FISH analysis was performed according to the manufactures'sprotocols using following probes for chromosome 5 and 8: a two-colorprobe for chromosome 5p15 (hTERT) and 5q35 (NSD1, Kreatech, Amsterdam,NL) as well as a three-color break apart probe for the chromosome 8q24(MYC, Kreatech, Amsterdam, NL). Evaluation of the hybridization signalswas done on an automatic spot counting system (Applied Spectral Imaging,Edingen/Neckarhausen, Germany). For each probe >300 nuclei were scannedand classified using a threshold of 5%.

Documentation

Prior to generation of the MSC-cell bank, the clinical specimen fortesting and the clinical specimen for release, the process had beenformally validated in small-scale cultures, based on which manufacturinginstructions (SOPs), batch records, testing instructions and protocols,as well as specifications were formally defined. A manufacturing licensefor a MSC-cell bank as well as for clinical specimen for use in clinicaltrials was obtained from the state government. Quality specificationswere set after formal advisories obtained from the Federal drug agency,the Paul Ehrlich Institute.

Statistical Analysis

Statistical significance was analyzed using GraphPad Prism 5 software(GraphPad Software, San Diego, Calif., USA). Significance was assessedusing the Student's t test. A P value less than 0.05 was consideredstatistically significant.

Example 1 Collection of Bone Marrow from 8 Healthy Third-Party Donorsand Isolation of BM-MNCs

After obtaining a written informed consent, from each bone marrow donorwere collected up to 250 ml additional bone marrow aspirate for thepurpose of MSC banking with approval by the local Ethics Committee infull agreement with the Declaration of Helsinki. In total from 8 donorsthe inventors obtained 1.66 liters bone marrow. For isolation of bonemarrow mononuclear cells by Ficoll-gradient the inventors used the Sepaxmachine as shown in FIG. 1. The absolute number of BM-MNCs per 1 ml ofbone marrow after this isolation procedure was 3.3×10⁶±6.3×10⁵ cells.Total number of BM-MNCs which was obtained from eight donors after twowashing steps was 9.86×10⁹. These cells were resuspended in cryomediumand distributed in the bags, that were frozen using a rate-controlledfreezer and then stored in the vapour phase of liquid nitrogen untiluse.

Example 2 Generation of Mesenchymal Stromal Cells from Bone MarrowMononuclear Cells-Establishment of the MSC-Bank

To generate the MSC-Bank, bone marrow mononuclear cells from 8 donorswere thawed, washed and pooled in DMEM supplemented with 5% PL. To findout the optimal concentration of platelet lysate for the adherence ofprogenitor cells of MSCs the inventors cultured BM-MNCs with bothconcentrations of PL: 5% and 10%. The obtained results have demonstratedthat the 5% concentration of platelet lysate is much more efficacious inpromotion of BM-MNCs and generation of MSCs than 10% concentration of PL(FIG. 2A). In addition, the inventors asked which of these twoconcentrations of PLs is better for clinical-scale expansion of MSCs.The inventors found that that the 10% concentration of PLs issignificantly more efficient in expanding the MSCs than the 5% PL (FIG.2B). Moreover, in both cases the unfiltered platelet lysates were moreeffective for generation and expansion of MSCs than the filtered ones(FIG. 2C). These preliminary experiments paved the way for establishingthe master MSC-bank. Therefore, the inventors thawed the BM-MNCs fromeach donor and after washing twice they were pooled together andthereafter cultured for 14 days, as described in the section of methods.The inventors were able to generate from 9.89×10⁹ BM-MNCs 3.2×10⁸ MSCsof passage 1. These MSCs expressed the typical markers for MSCs, such asCD73, CD90 and CD105 but were negative for hematopoietic cell markerse.g. CD14, CD34, CD45. They did not express HLA-Class II antigens butexpressed high levels of HLA-Class I antigens. According to trypan bluestaining the viability of these MSCs before freezing was 95±5%.

The total number of MSCs was distributed in 210 cryovials eachcontaining 1.5×10e6 MSC P1 and finally frozen in the gaseous phase ofliquid nitrogen until use. The inventors referred to this set of vialsas MSC-bank.

Example 3 Generation and Validation of the Clinical-Scale MSC-EndProducts

a) Thawing of Vials from MSC-Bank

To generate and test clinical-scale MSC-end products concerning theirproliferative, differentiation and allosuppressive potential, theinventors thawed three randomly chosen MSC-vials from the inventor'sbank 6-8 weeks after their cryopreservation. The cell mean averagerecovery from three thawed vials was 1.39×10⁶ viable cells/vial (range1.23×10⁶ to 1,48×10⁶), whereas the viability was 95, 25% (range 93,45%-96.9%). In average, expansion of these MSCs over 2 weeks until theend of passage 2 resulted in generation of 470×10⁶ viable MSCs (range420-548×10⁶ MSCs). These samples were frozen in bags until use andreferred to as clinical-scale MSC-end products.

b) Immunophenotyping of MSC-End Product and Their DifferentiationPotential

MSCs of the clinical end product at the end of passage 2 were negativefor the hematopoietic markers CD45, CD14, CD34 and did not expressHLA-DR. However, they expressed high levels of the typical MSC-markerslike CD73, CD90 and CD105. They were also able to differentiate alongosteoblasts and adipocytes in the tissue-specific media (FIG. 4).

d) Proliferation Kinetics and Senescence of MSCs

To demonstrate the rationale of pooling the bone marrow mononuclearcells from 8 donors in order to establish the inventor's MSC-bank, theinventors compared in vitro growth of MSCs from 8 individual donors withthe growth of pooled MSCs from each donor within P2 and 4 MSC-endproducts within the same passage (FIG. 5A). As expected, the MSCs ofeach bone marrow donor showed different growth kinetics varying from0.3×10⁶ (donor 7) to 1.7×10⁶ MSCs (donor 5). The mean proliferationkinetics of the MSC-product generated from pooled BM-MNCs of 8 donorswas 1.0×10⁶±0.5×10⁶ MSCs, which correlated astonishingly good with thenumber of MSCs generated from the pool of individual MSCs of 8 donors:1.1×10⁶. More interestingly, both values correlated very well with themean number of MSCs obtained from the expansion of 4 MSC-end productswithin a passage: 1.085×10⁶ ±0.1×10⁶ MSCs. These results, proved theinventor's assumption that by pooling the BM-MNCs it is possible togenerate an “arithmetical mean” of good and poorly proliferating MSCs.

In order to test the expectation that each MSC-end product from theMSC-bank possesses nearly the same proliferation potential the inventorsanalyzed it in expanded ten aliquots of the MSC-bank from PO to the endof passage 2 for clinical application. As shown in FIG. 5B the mean cellnumber of all expanded end products at the end of passage 2 was5.3×10⁸±5×10⁷ MSCs, indicating a highly homogeneous proliferationpotential of the end products. Likewise, the number of populationdoublings in P1 and at the end of passage 2 was rather the same (4.3PDs/passage) and cumulative number of PDs did not exceed the value 9(8.7±0.4). To test that MSCs are not immortal the inventors expanded 3MSC-end products from the inventor's MSC-bank over 12 passages. As shownin FIG. 5D, from passage 5 to 12 the MSCs undergo replicative senescenceand the number of PDs was promptly diminishing, indicating that thesecells are indeed senescent and do not proliferate indefinitely.

e) Allosuppressive Potential of MSCs Isolated from Individual Donors andMSC-End Products in Mixed Lymphocyte Reaction (MLR)

MSCs have been shown to exert allosuppressive properties either in vitroor in vivo. To test the inventor's assumption that MSCs generated fromthe pool of BM-MNCs of 8 donors may have a higher allosuppressivepotential than the average allosuppressive potential of MSCs generatedfrom individual donors, the inventors used in MLR the expanded MSCs ofpassage 2 from 8 individual donors as well as the MSC-pool that wasgenerated by pooling the MSCs of 8 donors before expansion (MSC-Pool),and one MSC-end product (generated from the MNC-pool derived MSC-bank:MSC-140). As expected, the allosuppressive potential of indiidual MSCswas very heterogeneous, i.e. these MSCs inhibited quite differently thealloantigen-induced proliferation of blood MNCs from two HLA-disparatedonors. This effect ranged from 20% (donors 1 and 8) to about 80%inhibition (donors 2 and 3) (FIG. 6A). The allosuppressive potential ofMSC generated from the pool of MSCs from 8 donors (MSC-Pool) was equalto the mean inhibitory potential of MSCs from 8 donors together (mean of8 donors). However, the allosuppressive potential of the expandedMSC-140 sample from the inventor's MSC-bank was significantly higherthan that of MSC-Pool and the mean allosuppressive potential of MSCsfrom 8 donors together (P<0.001, P<0.01, respectively). In order toassess whether the inventor's MSC-clinical products after thawing arehomogeneous in suppressing the alloreaction, the inventors thawed 6back-up vials with MSCs and directly tested in MLR assay, as they areadministred to patients in vivo. As shown in the FIG. 6B all these 6clinical MSC-products demonstrated a constant allosuppressive effect invitro, indicating their very homogeneous potential in suppressing thealloreaction. The mean allosuppressive potential at the target-effectorratios used here was 52%±8.7%.

Example 4 Genetic Characterization of the Clinical-Grade MSC-EndProducts

As in vitro culture may usually be the source of chromosomal aberrationsof cultured cells, the inventors asked whether the inventor'sclinical-grade MSC-end products are subject to such changes. Chromosomalanalysis of 25 MSC-mitoses with a resolution of approximately 350-400bands demonstrated a normal number of chromosomes (euploidy) in all ofthem (FIG. 7A). However, by using a resolution of approximately 300bands the inventors found a translocation between the short arm ofchromosome 5 and the short arm of chromosome 9 in 4 out of 25 analyzedmitosis. The breakdown points were localized in the band 5p13 and19p13.3. Fluorescent in situ hybridization (FISH) analysis using atwo-color probe for chromosome 5p15 (hTERT) and 5q35 (NSD1) as well as athree-color break apart probe for the chromosome 8q24 demonstrated thatthe majority of clinical-grade MSC-end products from the inventor'sMSC-bank possess a normal diploid pattern for both chromosomes (FIG. 7B,C). Interphase nuclei after two-color hybridization of probe set 5p15(green) and 5q35 (red) identified that 97.2% of cells demonstrated anormal diploid pattern for chromosome 5 and only about 2.8% showed atetraploid hybridization pattern (FIG. 7D). Likewise, interphase nucleiafter three-color hybridization of MYC break apart probe (FIG. 7C)showed in 97% of MSCs two normal fusion signals for chromosome 8q24 and3% of MSCs with a tetraploid signal pattern (FIG. 7E). Thus, a verysmall fraction of MSCs acquire chromosomal aberrations in vitro.

Analysis of p53, p21 and Myc gene expression in 3 clinical-scale MSC-endproducts demonstrated a 2 to 5-fold increase in expression of p21, anabout 6 to 10-fold reduction of p53 gene expression and no expression ofthe proto-oncogene c-myc (FIG. 8A). Most importantly, in consent withthe senescent behaviour of MSCs from the inventor's bank, the inventorsdemonstrated that none of the 3 MSC-end products expressed hTERT (datanot shown).

As the MSCs of the inventor's bank were generated from the pool ofBM-MNCs of 8 third-party donors, the inventors were interested in therelative contribution of each donor after generation of the MSCs.Chimeric analysis by STR-PCR using a series of genetic markersdemonstrated the presence of 8 donors in different proportions withinthe clinical-scale MSC-end product (FIG. 8B). In principle, thepercentage of presence in the clinical product correlated with theproliferation potential of MSCs generated from individual donors i.e.MSCs that individually expanded better were also found in higherproportions in the MSC-end product.

Example 5 Clinical Cases of Patients Treated with MSC Preparations ofthe Invention

Patient 1 born Mar. 26, 1999

Disorder: Thalassemia Major

After receiving stem cell transplantation the patient developed ascites,a swelling of the joints, pericardial effusion, caused by animmunological polyserositis, possibly in the context of graft versushost disease (GvHD). The administration of MSC of the invention onceproceeded without complications. Under concomitant treatment with adiuretic the ascites, the swelling of the joints and pericardialeffusion disappeared.

Patient 2 born Dec. 20, 2009

Disorder: Severe Agranulocytosis

On day+12 after stem cell therapy (SCT), the patient showed an acutefoudroyante GvHD of the skin (grade IV), which even with 2 mg/kg steroidand 55 mg/kg Mofetilmycophenolat daily was uncontrollable. On Nov. 22,2012 the child received the inventive MSC.

After MSC therapy the GvHD slowly but constantly declined, until itcould no longer be detected after day 28 of MSC administration. Thechild has tolerated the MSCs very well and showed no viral, bacterial orfungal infection in the 30 days after administration.

Patient 3 born Mar. 25, 2010

Disorder: Acute Hymphoblastic Leukemia;

SCT was on 16.10.12 from an HLA-non-identical, related, donor. Alreadyon day +14 after SCT, the first signs of GvHD in the intestine werenoted. Immediate therapy with Mofetilmy-cophenolat and steroid wasstarted. That led to a relative improvement in symptoms. On day +35after SCT, however, an intestinal GvHD grade II remained in spite oflong-term steroid administration.

The decision was therefore taken to consolidate the immunosuppressivetherapy by the administration of the inventive MSCs. The MSCadministration took place without complications on Dec. 21, 2012. Noinfections showed up in the 30 days after SCT administration. On15.1.2013 no signs of intestinal GvHD in the patient could be observed,and only a mild GvHD of the skin that did not require any treatmentremained.

Patient 4 born Mar. 25, 1999

Disorder: Acute Myeloid Leukemia;

SCT on Dec. 19, 2012 from an HLA—Non-Identical, Related Donor

5 months after stem cell transplantation, the patient developed clinicalsymptoms of Stevens-Johnson syndrome. Since the patient at the time ofrecording took several medications that are associated with thedevelopment of this clinical picture, the respective medication wasended (voriconazole, penicillin, co-trimoxazole). Due to the detectionof an adenovirus infection, it was decided not to conduct aimmunosuppressive therapy with glucocorticoids. The patient developedalso typical GvHD skin lesions that were probably stimulated by theStevens-Johnson syndrome. Therefore, the immunosuppressive therapy withCSA was initiated. In order to dampen the inflammatory events, MSCs ofthe invention were administered. This MSC administration was welltolerated. Also a glucocorticoid - containing cream was administeredseveral times a day. Under this therapy, there was an almost completehealing of the GvHD skin lesions.

1. An in vitro method for the isolation of mesenchymal stromal cells(MSC), comprising the steps of: a) pooling individual bone marrowsamples obtained from at least two genetically distinct donors to obtaina sample cell-pool; and b) isolating mesenchymal stromal cells from saidsample cell-pool.
 2. The method according to claim 1, further includingthe step: culturing said sample cell-pool prior to, isolatingmesenchymal stromal cells from said sample cell-pool.
 3. The method ofclaim 1, wherein said bone marrow sample is a mammalian bone marrowsample and said mesenchymal stromal cell is a mammalian mesenchymalstromal cell.
 4. The method according to claim 1, wherein said bonemarrow samples are bone marrow mononuclear cell samples.
 5. The methodaccording to claim 1, wherein said bone marrow samples are obtained fromat least three, at least four, at least five, at least six, at leastseven, or at least eight genetically distinct donors.
 6. The methodaccording to claim 1, further including the step of storing saidisolated mesenchymal stromal cells.
 7. The method of claim 1, furtherincluding the steps of: a) determining the hTERT negative and polygenicstatus of the isolated mesenchymal stromal cells; and b) enriching theisolated mesenchymal stromal cells to obtain hTERT negative andpolygenic isolated mesenchymal stromal cells.
 8. The method of claim 7,further including the step of enriching the hTERT and polygenicisolated, enriched mesenchymal stromal cells to: a) At least 95% CD73+cells, and/or b) At least 95% CD90+ cells, and/or c) At least 95% CD105+ cells, and/or d) At least 95% HLA-class 1+ cells, and/or e) Lessthan 1% CD45+ cells, and/or f) Less than 1% CD 14+ cells, and/or g) Lessthan 1% CD34+ cells, and/or h) Less than 5% HLA-DR+ cells.
 9. The methodof claim 8, wherein the hTERT and polygenic isolated mesenchymal stromalcells are (a), (b) and (c), and/or (e), (f) and (g).
 10. (canceled) 11.An isolated cellular composition comprising bone marrow cells from atleast two genetically distinct bone marrow donors, made by a processcomprising the steps of: a) pooling at least two monogenic andgenetically distinct bone marrow samples to obtain a sample cell-pool;and b) isolating a stromal cell fraction from the sample cell-pool tothereby obtain the isolated cellular composition.
 12. The isolatedcellular composition of claim 11, further including the step ofisolating mesenchymal stromal cells from the isolated cellularcomposition to thereby obtain isolated mesenchymal stromal cells. 13.The isolated cellular composition of claim 12 for use in a method oftreating an autoimmune disease in a subject in need of treatment. 14.The isolated cellular composition of claim 13, wherein the autoimmunedisease is multiple sclerosis, Type 1 diabetes, rheumatoid arthritis,uveitis, autoimmune thyroid disease, inflammatory bowel disease (IBD),scleroderma, Graves' Disease, lupus, Crohn's disease, autoimmunelymphoproliferative disease (ALPS), demyelinating disease, autoimmuneencephalomyelitis, autoimmune gastritis (AIG), autoimmune glomerulardiseases, and preferably or graft-versus-host disease (GvHD).
 15. Themethod of claim 3, wherein the mammalian bone marrow sample is a humanbone marrow sample and the mammalian mesenchymal stromal cell is a humanmesenchymal stromal cell.
 16. The method of claim 1, further includingthe step of culturing the isolated mesenchymal stromal cells.
 17. Themethod of claim 1, further including the step of administering theisolated mesenchymal stromal cells to a subject in need of treatment fora disease.
 18. The method of claim 17, wherein the isolated mesenchymalstromal cells that are administered to the subject are administered totreat an autoimmune disease.
 19. The method of claim 18 wherein theautoimmune disease is multiple sclerosis, Type 1 diabetes, rheumatoidarthritis, uveitis, autoimmune thyroid disease, inflammatory boweldisease (IBD), scleroderma, Graves' Disease, lupus, Crohn's disease,autoimmune lymphoproliferative disease (ALPS), demyelinating disease,autoimmune encephalomyelitis, autoimmune gastritis (AIG), autoimmuneglomerular diseases, and graft-versus-host disease (GvHD).
 20. Themethod of claim 19, wherein the autoimmune disease is graft-versus-hostdisease.
 21. An isolated mesenchymal stromal cell preparation comprisingat least two genetically distinct hTERT negative and polygenicmesenchymal stromal cells.
 22. The isolated mesenchymal stromal cellpreparation of claim 21, wherein the hTERT negative and polygenicmesenchymal stromal cells are: a) At least 95% CD73+ cells, and/or b) Atleast 95% CD90+ cells, and/or c) At least 95% CD 105+ cells, and/or d)At least 95% HLA-class 1+ cells, and/or e) Less than 1% CD45+ cells,and/or f) Less than 1% CD 14+ cells, and/or g) Less than 1% CD34+ cells,and/or h) Less than 5% HLA-DR+ cells.
 23. The isolated mesenchymalstromal cell preparation of claim 22, wherein the hTERT negative andpolygenic mesenchymal stromal cells are (a), (b) and (c), and/or (e),(f) and (g).